Invasive cervical cancer (ICC) is the fifth most common malignancy in the world, accounting for 400,000 cases per year. A number of independent risk factors have been identified of which infection by human papillomavirus (HPV) is the most consistent. Eighty to 90 percent of ICCs are virus-positive. However, HPV infection alone is not sufficient for the development of cervical cancer since in some populations up to 70 percent of women are infected yet the malignancy develops in less than 8 percent. Therefore, genetic changes must play a role in tumor development. In preliminary experiments we show that 50 percent of all tumors tested have lost all or part of the short arm of human chromosome 6 suggesting that a gene, important for the development of ICC, is located there. Our latest data allowed us to narrow down the region to 400 kb that is almost completely sequenced by the Sanger Center. The aim of this proposal is to identify the gene in 6pZ3 that causes ICC. Transcripts found within the candidate region will be identified from the sequence by either experimental or computer-based methods. In the latter category we will also perform homology-based analysis with the mouse genomic sequence that is being generated for this project. We already found 2 to 4 genes in the available sequence. All candidate genes will be characterized for their pattern of expression. Genes expressed in cervical epithelium will be screened for mutations in the tumors with 6p deletions. For mutation screening we will obtain data on each gene's structure, including its full-length sequence and the intron/exon junctions. We will screen either deleted tumor RNAs or DNAs for mutations by DHPLC and direct sequencing of PCR products. Once the tumor suppressor gene is identified, our extensive cervical tumor bank will be screened for mutations and the results correlated to clinical and pathologic factors. In addition, experiments aimed at the determination of the gene's function such as its regulation during cell cycle, interaction with other cell cycle controlled genes etc., will be initiated. Identification of a gene important in cervical carcinogenesis will allow the development of better diagnostic and prognostic markers to reduce the morbidity and mortality from this cancer.